The generation of an immune response against a pathogen (bacterial, viral or parasite) depends, in the first instance, on the delivery of the appropriate stimulus to the immune system of the host. The pathogen or infectious agent presents the host with a number of immune-stimulating compounds or antigens which are usually large molecules such as proteins, polysaccharides or glycoproteins. These antigens may provoke one or more different types of reaction from the host in an effort to destroy or eliminate the invading organism. Accordingly, the antigen may stimulate T-cells which provide cell-mediated immunity and/or an antigen may stimulate B cells to initiate the synthesis and secretion of antibody (humoral immunity). The development and maintenance of the individual's protective immune response to a foreign antigen is usually dependent on achieving a critical level of stimulation of both cell-mediated and humoral immunity.
In the generation of a protective immune response, a certain type of T-cell, a helper T-cell is frequently required to assist the B-cell to grow and secrete soluble antibody. These helper T-cells also interact with and recognize antigens on the surface of antigen-presenting cells such as macrophages and, by releasing soluble factors (cytokines), mediate activation and differentiation of B-cells.
Certain small molecules termed haptens, of which short peptides are an example, are usually poorly immunogenic while larger molecules such as proteins and some polysaccharides are usually immunogenic in that they elicit a satisfactory protective response. To obviate the problems of inducing immunity to poorly immunogenic molecules, attempts have been made to enhance their immunogenicity by binding them to "carrier" molecules. These carriers, which are usually immunogenic proteins, function by stimulating the T-cell co-operative effect that occurs with naturally immunogenic molecules. That is to say, a poorly immunogenic antigen, bound to a carrier, will elicit T-cell help in antibody production. By engaging the T-cells with carrier determinants, B-cells will begin antibody production not only to the carrier itself, but also to the bound antigenic determinant.
Although it is widely accepted that the carrier principle is an effective method of improving the efficacy of vaccines, the number of proteins which are ethically accepted for use as potential carrier proteins for human use is relatively limited. These include tetanus toxoid and diphtheria toxoid. The limited number of available carrier proteins means that a large number of vaccine products will employ one of these proteins and multiple immunizations with products conjugated to these carriers increases the possibility that undesirable reactions to these carriers will occur. Also, these carriers have been chosen in the first instance, not for their immunostimulatory characteristics, but rather because they were already registered for human use. It is clear, therefore, that there is a need for an alternative carrier to those currently used in conjugate vaccines which will obviate the immunological problems associated with these vaccines and yet retain the same immunogenicity as the vaccines presently in use or improve on it.
During the past decade it has become clear that certain fragments of proteins, rather than the entire protein molecule, are preferentially recognized by T-cells in association with an appropriate self (Class I or Class II) antigen. These fragments are known as T-cell epitopes and their co-recognition (i.e., in association with certain Class I or Class II molecules) by T-cells ensures the delivery of "T-cell help" so that a B cell can be activated and undergo differentiation to secrete antibody.
It is generally accepted that T-cell recognition of proteins is more complex than antibody binding, and, despite recent advances in our understanding of T-cell epitopes, less clearly understood. However, in the mid 1980s it was suggested that T-cell determinants (epitopes) have a tendency to form stable helical structures in which the hydrophilic groups align on one surface of the helix while hydrophobic residues align on the opposing surface. In this model, it is proposed that the hydrophobic surface would normally be found associated with the MHC antigen while the more hydrophilic surface would be exposed to the T-cell receptor. Accordingly, an algorithm to search a given protein sequence for regions with a tendency to form helical amphipathic structures has been developed and applied to several protein models (De Lisi and Berzofsky, P N A S 82: 7048, 1985). In contrast, some workers maintain that T-cell determinants are associated with beta turns within the protein. However, these algorithms frequently fail to detect T-cell epitopes and conversely often select sequences which do not function as T-cell epitopes. In addition, these algorithms can not be used to define the strength or cross-species functionality of selected sequences. A unifying hypothesis of what factors are important for predicting T-cell epitopes has yet to emerge and the identification of such epitopes as well as the determination of their strength is still very much an empirical exercise. Although it is still not clear what a
T-cell perceives, there is agreement among several groups using a variety of models that a region of 7-17 amino acid residues in length is required for recognition.
T-cell epitopes from diphtheria toxin, tetanus toxin and cross-reacting material of diphtheria toxin were described in PCT/US89/00388. They differ from the T-cell epitopes of this invention.
Previous work (PCT/AU87/00107) has examined a number of integral membrane proteins for their ability to generate serum antibody responses in the absence of adjuvant. These proteins, which include TraT, have been shown to stimulate high titres of serum antibody in mice, rats, guinea-pigs and rabbits. The antibody titres elicited by injecting TraT in saline is not significantly increased by the addition of oil-based adjuvants such as Freund's Incomplete Adjuvant (FIA) or MONTANIDE/MALCOL. Covalent attachment of Bovine Serum Albumin or of the dinitrophenyl group or of a peptide antigen to TraT results in a significant enhancement of the immune response to the conjugated material as compared with the response seen when the immunogen is injected without adjuvant or not conjugated to TraT. The antibody response to these conjugates is not significantly increased by the addition of FIA. TraT is a self-adjuvanting carrier molecule which is capable of generating high antibody titres to itself as well as to molecules attached to it.